Standardized uptake value (SUV) normalized by lean body mass ([LBM] SUL) is recommended as metric by PERCIST 1.0. The James predictive equation (PE) is a frequently used formula for LBM estimation, but may cause substantial error for an individual. The purpose of this study was to introduce a novel and reliable method for estimating LBM by limited-coverage (LC) CT images from PET/CT examinations and test its validity, then to analyse whether SUV normalised by LC-based LBM could change the PERCIST 1.0 response classifications, based on LBM estimated by the James PE.LC) and whole-body fat mass (FMWB). This equation was cross-validated with an independent sample of 97 patients who also received whole-body PET/CT examinations. Its results were compared with the measurement of LBM from whole-body CT (reference standard) and the results of the James PE. Then, 241 patients with solid tumours who underwent PET/CT examinations before and after treatment were retrospectively retrieved. The treatment responses were evaluated according to the PE-based and LC-based PERCIST 1.0. Concordance between them was assessed using Cohen’s κ coefficient and Wilcoxon’s signed-ranks test. The impact of differing LBM algorithms on PERCIST 1.0 classification was evaluated.First, 199 patients who received whole-body PET/CT examinations were retrospectively retrieved. A patient-specific LBM equation was developed based on the relationship between LC fat volumes (FVLC were significantly correlated with the FMWB (r=0.977). Furthermore, the results of LBM measurement evaluated with LC images were much closer to the reference standard than those obtained by the James PE. The PE-based and LC-based PERCIST 1.0 classifications were discordant in 27 patients . These discordant patients’ percentage changes of peak SUL (SULpeak) were all in the interval above or below 10% from the threshold (±30%), accounting for 43.5% (27/62) of total patients in this region. The degree of variability is related to changes in LBM before and after treatment.The FVpeak close to the threshold.LBM algorithm-dependent variability in PERCIST 1.0 classification is a notable issue. SUV normalised by LC-based LBM could change PERCIST 1.0 response classifications based on LBM estimated by the James PE, especially for patients with a percentage variation of SUL Fundamereatment . To ensureatment .Nevertheless, there are still many sources of error in SUV measurement . PERCISTComputed tomography (CT) has become a standard method for measuring body composition . BecauseIntuitively, more accurate measurement of LBM estimates from LC images should produce more accurate SUL normalisations than LBM estimates from the James PE. Narita et al.  reported18F-FDG PET/CT examinations between December 2011 and March 2018 were retrospectively reviewed. All patients were randomly assigned into equation-development (106 men and 93 women) and validation (54 men and 43 women) groups. Then, patients with solid tumours who underwent 18F-FDG PET/CT before and after treatment were retrospectively retrieved as the application group between the same periods. The inclusion criteria of this group were as follows: non-diabetic, still-existing 18F-FDG-avid lesions and no appearance of new 18F-FDG-avid lesions after treatment. Finally, a total of 241 patients (145 men and 96 women) were included in this study, including 72 with non–small cell lung cancer, 53 with hand and neck tumours, 46 with breast cancer, 22 with oesophageal cancer, and 48 with colorectal cancer. The clinic variables of patients were recorded, which included sex, age, height, weight, body-mass index (BMI), blood glucose level, injected dose of 18F-FDG, and uptake time. The study was approved by the institutional review board of the First Affiliated Hospital of Jinan University and complied with national legislation and the Declaration of Helsinki guidelines. All patients gave consent to use their 18F-FDG PET/CT results and relevant clinical data for this study.First, a total of 296 patients who received whole-body 18F-FDG PET/CT examinations were performed with the same protocol using a GE Discovery PET/CT 690 system. PET/CT images were acquired 50 to 70 min after intravenous injection of 18F-FDG at a dose of 0.08–0.10 mCi/kg body weight. The scanning range covered the whole-body for the equation-development and validation groups and covered from the top of the skull to mid-thigh for the application group. CT data were acquired in breath-hold with 120 kV, 80–160 mA modulated using the GE AutomA technique with a slice thickness of 3.75 mm, slice interval of 3.27 mm, pitch of 1.375, matrix size of 512 × 512 and scan field-of-view of 50 cm. PET data were acquired in 3D time-of-flight (TOF) mode with a 2-min scan per bed position, overlap of 23.4%, slice thickness of 3.27 mm, slice interval of 3.75 mm, pixel size of 3.64 mm, matrix size of 192 × 192 and scan field-of-view of 70 cm. The PET data were reconstructed in terms of the point spread function (PSF) together with TOF technology.All WB) [LC) were measured using the same method.Fat tissues were defined as voxels identified and measured by CT as having CT numbers between −190 and −30 Hounsfield units. A built-in software package of the Advantage Workstation  was used to calculate fat volumes (FV). A fat-tissue average density of 0.923g/mL was applied to convert whole-body FV to whole-body fat mass (FMWB) . LC imagWB cannot be measured in conventional PET/CT examination, the relationships between FMWB and FVLC were analysed in the populations from the equation-development group, and an equation was developed using FMWB as the dependent variable and FVLC as independent variables. Subsequently, the LBM could be estimated from LC images (LBMLC) according to the following equation:Traditionally, LBM was equivalent to the fat free mass (FFM) . Becauseα and β are the intercept and slope of the equation we developed, respectively.where To test the reliability of this method, LBM by LC images was compared with those derived by the James PE in an independent sample of 97 patients from the validation group. The reference standard was the measurement of LBM from whole-body CT. The James PE was defined as follows:W is weight in kilograms, and H is height in centimeters.where peak) of target lesion (SULpeak-PE), is one program the clinician can use to assist in monitoring treatment response according to PERCIST 1.0. This software can use an iterative adaptive segmentation algorithm to find a threshold value that separated the target volume from the background tissue by weighting the maximum SUV (SUVmax) and mean SUV (SUVmean) within the target volume with a default weighting factor of 0.5. A volume of interest (VOI) is placed over the tumor, and then, the software automatically measures the SULpeak-PE within the entire tumor. Adjustment of the estimated tumor surface was sometimes needed to include the entire tumor within the margins of the volume of interest. The detailed instructions for PET VCAR were described in our previous study [PE calculated by Eq. peak-PE of target lesion measured by PET VCAR, the LC image-based SULpeak (SULpeak-LC) of the same target lesion could be calculated.The therapeutic responses were analysed with PET volume computer-assisted reading (PET VCAR) of the Advantage Workstation . PET VCAR, using the James PE to estimate LBM and then measuring the peak SUL , stable metabolic disease (SMD), and progressive metabolic disease (PMD) separately according to PERCIST 1.0.As defined in PERCIST 1.0 , the nort test was used to analyse the differences in patients’ characteristics between the equation-development and validation groups. For the equation-development group, Pearson correlation coefficients (r) were used to evaluate the relationship between the FVLC and FMWB. Then, simple linear regression was applied to generate equation to estimate FMWB from FVLC. The populations from the validation group were used for cross-validation: paired t test and Bland-Altman plots were used separately to examine the difference and agreement between the outcomes of LBMPE and LBMLC and the reference standard.The patients’ characteristics were presented as mean values and standard deviations. The unpaired t test was used to evaluate the differences in the same parameters between before and after treatment for the application group. Concordance and differences among the PERCIST 1.0 results of these two methods were assessed using Cohen’s κ coefficient and Wilcoxon’s signed-ranks test. Furthermore, to evaluate the impact of the LBM algorithm on assessment of the therapeutic response, we first analysed the distribution of SULpeak-PE variation in patients with discordant response classifications. Subsequently, the variation of the LBMPE and LBMLC was evaluated in these patients. Graphs and analyses were performed using Prism GraphPad and the SPSS software.The paired P>0.05). For the application group, there were no significant differences in blood glucose levels, injected doses of 18F-FDG, and uptake times between baseline and follow-up examinations (P>0.05). The interval between both PET/CT examinations was 136±51 days.Patients’ clinical characteristics and parameters in the equation-development, validation and application groups are shown in Table LC were 11084.01 cm3 (range of 1362.69–31516.25 cm3) and the mean FMWB was 17.71 kg (range of 2.55–45.48 kg). There was a strong correlation between them . According to this relationship, a patient-specific equation for LBM from FVLC was established:For patients from the equation-development group, the mean FVPE, LBMLC and LBMWB were 47.01 ± 8.38, 42.26 ± 8.23, and 42.13 ± 8.43 kg, respectively. The LBMPE results were significantly higher than the LBMWB results (P<0.001), according to the paired t test, whereas the difference was not statistically significant between the LBMLC and LBMWB (P=0.408). Bland–Altman plots for each of estimated LBMs are shown in Fig. PE, LBMLC exhibited stronger agreement with LBMWB, with closer mean (−0.1 kg) to LBMWB mean and narrower 95 % CI .For patients from the validation group, the results of LBMpeak was –39.78% ± 18.52 and +34.03% ± 19.18, respectively, in the groups of tumours showing a decrease and an increase in 18F-FDG uptake, with 47 patients showing PMD, 82 SMD and 112 PMR. With LC image-based PERCIST 1.0, the percentage change in SULpeak was –39.07% ± 17.88 and +34.19% ± 18.85, respectively, in the groups of tumours showing a decrease and an increase in 18F-FDG uptake, with 53 patients showing PMD, 75 SMD and 113 PMR. PE-based and LC image-based PERCIST 1.0 classifications were discordant in 27 patients (11.2 %), with an almost perfect agreement in response classification between the two assessments . Over 10 %, although the variations of SULpeak-LC were different with SULpeak-PE, these differences were not enough to change the original classifications. For all patients from the application group, a total of 62 patients’ percentage change of SULpeak-PE distributed in this interval.The distributions of percentage change in SULpeak-PE and SULpeak-LC are shown in Fig. peak-PE were found in the interval above or below 5% from the threshold, and 17 (70.8 %) discordances were found in this region. In addition, 38 patients’ PE-based SULpeak were found in the interval above or below 5–10% from the threshold, and 10 (26.3%) discordances were found in this region.The percentage change of 62 patients’ SULPE (7.2% ± 2.2) were significantly higher than those of LBMLC (2.8% ± 1.8) (P<0.001). Five patients’ LBM showed increase, resulting in changes from SMD to PMR, and PMD to SMD. The increase percentages of LBMPE (5.7% ± 1.5) were significantly higher than those of LBMLC (1.8% ± 1.7) (P=0.001). Furthermore, six patients’ weight increase and therefore LBMPE showed increase (1.8% ± 1.0) at the follow-up examination, whereas LBMLC showed decrease (4.8% ± 1.9), resulting in changes from SMD to PMR, and PMD to SMD.Of 27 patients with discordant response classifications, 16 patients’ LBM showed a decrease at the follow-up examination, leading to PMR being changed to SMD and SMD to PMD. The reduction percentages of LBMpeak-PE could be reclassified according to the SULpeak-LC. The closer the percentage changes of SULpeak to the threshold, the greater the change that could have happened.In this study, we proposed a novel and reliable method to estimate the patient’s whole-body LBM from the LC images, and the results of this method showed an excellent agreement with the LBM obtained from the whole-body CT. Furthermore, and more importantly, we found that the classifications of PERCIST 1.0 according to the SULr=0.88–0.963) [LC were significantly correlated with the FMWB (r=0.977). Compared with single-slice, the region of LC contained more fat tissues in the body and theoretically could enable estimation of FMWB more accurately.CT technology is considered one of the preferred approaches for measuring FM , but the8–0.963) –19. Acco8–0.963) . In thisPE was significantly higher than the reference standard, but not for the LBMLC. The Bland–Altman plots showed that the results of LBMLC were more accurate than those of LBMPE.Previous studies demonstrated that the LBM from the LC images was more accurate than the results obtained from the James PE. In a study by Chan , the LBMPE is that the LBM has ethnicity specificity [LC and FMWB to estimate LBM. The reason for selecting the region of the thorax to ischium is that the conventional PET/CT scans definitely included this anatomic region, and the anatomical location is clear and easy to popularise.A reasonable explanation for the heterogeneity of LBMcificity , the Jamcificity . On the cificity , who estpeak-PE and SULpeak-LC and, more important, to the variance in LBM between the baseline and follow-up scans. In general, patients who received cancer treatment suffered from weight loss, especially the skeletal muscle loss [PE was significantly higher than that of LBMLC (P<0.001), and the percentage change of SULpeak-PE was therefore smaller than that of SULpeak-LC. These could lead to worse classifications when the percentage change of SULpeak-LC exceeded the thresholds. In addition, five of 27 patients had an increase in LBM after treatment; conversely, the response evaluation based on LC image-based PERCIST 1.0 tended to be more optimistic, from SMD and PMD reclassified as PMR and SMD.Through the clinical application, we found that PE-based and LC image-based PERCIST 1.0 was discordant in 27 (11.2 %) patients. These discordances were due to differences in the values of SULcle loss , 24. In PE after treatment, but LBMLC showed a decrease, resulting in more optimistic classifications based on LC images-based PERCIST 1.0. These discrepancies are most likely explained by a state, for patients with chronic diseases, in which the loss of muscle mass was associated with preserved or even increased body fat content [It is noteworthy that six of 27 patients had an increase in body weight and therefore in LBM content . Multipl content , 26. The18F-FDG PET imaging conditions and provides a much more detailed framework for lesion selection, region of interest definition and response classification [Although heterogeneity was found only in the region close to the threshold, it is also crucial, because for individual patients, different response classifications may lead to different treatment programs. To ensure the accuracy of response evaluation, PERCIST 1.0 describes in detail methods for controlling the quality of fication . However2) included in the equation-development group. Whether this equation is suitable for obese patients needs further verification. Large subject samples with wide ranges of body mass and height are needed in the future study for equation development. Second, we only analyse the James PE which commonly incorporated in the modern PET/CT systems. Other formulas, especially the Janmahasatian PE [LC should produce more accurate PERCIST 1.0 classifications than LBMPE, further prospective study is needed to investigate the correlation of these discordances with pathological examinations to confirm this conclusion.The current study had some limitations. First, only 6 obese patients (BMI ≥ 30 kg/matian PE  which haatian PE , should peak close to the threshold. The degree of variability is related to changes in LBM before and after treatment.The whole-body LBM could be estimated from LC CT data acquired in PET/CT examinations. The patient-specific LBM was more accurate than results obtained by the James PE, which has been routinely used in the modern PET/CT system. LBM algorithm-dependent variability in PERCIST 1.0 classification is a notable issue. SUV normalised by LC images-based LBM could change PERCIST 1.0 response classifications based on LBM estimated by the James PE, especially for patients with a percentage change of SUL


In this study, using the proximity ligation assay, we assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. Our data demonstrates that ICAM-1 harboring HM or hybrid epitopes as well as ICAM-1 bearing α-2,6-sialylated epitopes are present in human and mouse atherosclerotic lesions. Further, HM-ICAM-1 positively associated with increased macrophage burden in lesions as assessed by CD68 staining, whereas α-2,6-sialylated ICAM-1 did not. Finally, both HM and α-2,6-sialylated ICAM-1 N-glycoforms were present in hemodialysis patients who had AVF maturation failure compared to successful AVF maturation. Collectively, these data provide evidence that HM- ICAM-1 N-glycoforms are present in vivo, and at levels similar to complex α-2,6-sialylated ICAM-1 underscoring the need to better understand their roles in modulating vascular inflammation.Endothelial dysfunction is a critical event in vascular inflammation characterized, in part, by elevated surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is heavily N-glycosylated, and like other surface proteins, it is largely presumed that fully processed, complex N-glycoforms are dominant. However, our recent studies suggest that hypoglycosylated or high mannose (HM)-ICAM-1 N-glycoforms are also expressed on the cell surface during endothelial dysfunction, and have higher affinity for monocyte adhesion and regulate outside-in endothelial signaling by different mechanisms. Whether different ICAM-1 N-glycoforms are expressed  Inflammation is a carefully orchestrated response involving the release of pro-inflammatory factors and homing of immune cells to injured tissue. The vascular endothelium is a key player in the inflammatory process, responding to stressors such as oscillatory blood flow (that occurs at bifurcations in the vessel) and increased pro-inflammatory factors –3. The rX ligands Reviewers' comments:Reviewer's Responses to Questions5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The study by Regal-McDonald et al used PLA to investigate co-localization of specific N-glycan structures and ICAM-1 in mouse and human atherosclerotic arteries and human AVF of chronic kidney disease patients . The results demonstrated that hypoglycosylated and α-2,6-sialylated ICAM-1 N-glycoforms are present in atherosclerotic murine and human arteries and AVF patients. The results also indicated that HM-ICAM-1 levels positively correlated with macrophage burden in both murine and human atherosclerotic tissue. The study is timely but lacks functional information on the role of ICAM-1 N-glycoforms in atherosclerosis or AVF.1. The figures of the manuscript are very low resolution.2. It would strengthen the findings of the manuscript if the authors confirm the results obtained using the innominate arteries by looking at total, HM / hybrid, a-2,6-sialylated, and a-2,3-sialylated ICAM-1 in the atherosclerotic mouse aorta.3. How were the lesion and non-lesion segments identified?4. Fig. 2 and 3 legends state that n=5 experiments were performed for CD68 immunostaining. The corresponding bar graphs show only 3 experiments.5. What is the correlation coefficient for Fig. 4 studies? It seems there would be no correlation if only the closed symbols are taken into consideration. It is recommended to perform more experiments for the correlation studies.In the high fat diet model , there was atherosclerosis in the brachiocephalic artery and HM/hybrid, HM, a-2,6-sialylated and a-2,3-sialylated ICAM-1 were increased in lesion vs. non-lesion segments. If there is atherosclerosis in these mice but no correlation between CD68 and ConA or HHL puncta, the mechanisms regulating ICAM N-glycosylation may not be important for monocyte adhesion and macrophage accumulation.6. Type 2 human atherosclerotic lesions show no ICAM-1 expression. The type 2 lesions are characterized by the presence of lipid-laden macrophages according to Stary et al . These findings suggest that ICAM-1 (and its N-glycoforms) does not play a role at the early phase of monocyte transmigration in human atherosclerotic vessels.7. Fig. 6B legends n=10 is misleading. It seems only n=5 samples per group was involved in the analysis. The authors need to carefully check the legends for accuracy.8. This is an observational study and no functional experiments have been included to investigate the role of different ICAM-1 N-Glycoforms in the development of atherosclerosis or pathogenesis of AVF maturation failure. The manuscript would be strengthened by the addition of mechanistic data.9. The rational for including the AVF data in the manuscript is not entirely clear. The mechanisms, leading to endothelial dysfunction in AVF failure vs atherosclerosis is different.Reviewer #2: This is a novel approach to assessment of ICAM-1 N-Glycoforms in mouse and human models of endothelial dysfunction. In this paper using the proximity ligation assay (PLA), authors assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis.To establish the distinct ICAM1-N-glycoforms in inflammatory disease in ivvivo authors solely used PLA assay. Indeed this assay has some limitation, however the PLA positive red puncta was not clear in all images throughout the manuscript. As this data represents the first study to demonstrating ICAM1-N-glycoforms invivo authors should provide convincing images with good quality pictures for PLA assay.There was no description for Figure 2F and 2G in result section. Author should mention about these two figures.In result section, Figure 3A author described that total ICAM-1, HM / hybrid N-glycoforms were increased in the ligated left carotid artery (LC) compared to the control right carotid artery (RC). But this statement was not matches with the Figure 3A. It looks like total ICAM1 and α-2,6-sialylated ICAM1 expressed more than HM/hybride. Fig 3C quantification was not reflects the Fig 3A images. Author should be careful about this discrepancy.In Fig 4 and Fig 6 show correlations between CD68 staining and HM / hybrid, HM and α-2,6- sialylated, ICAM-1 respectively. It was not clear how author made this correlation graph. Author should provide some direct evidence to prove this statement that CD68 expression higher in HM/hybrid not other ICAM1 N-glycoformes.Partial ligation and HFD are well establish model for atherosclerosis. Authors used only male mice without justification for the exclusion of female mice. Author should comment on this in discussion.Overall, this manuscript does not fit for publication in PLoS One as it currently stands.Reviewer #3: The current study showed correlation of high mannose (HM)-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms with macrophage burden in the lesions in human and mouse atherosclerosis, and in AVF maturation failure in hemodialysis patients. HM-ICAM1 N glycofoms and sialylated ICAM1 N-glycoforms were assessed by proximity ligation assay, while macrophage burden were assessed by CD68 staining. This is an extension of author’s previous studies. Findings are novel and important. There are several issues that should be addressed to make manuscript stronger.1) Overall, image quality except Figure 7A is poor. High resolution image will be needed, since this is a crux of current study.2) To support HM-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms in lesions, they need to be shown by western analysis as well.3) In panel 2F, 3D, 6A, please show macrophage staining by immunohistochemistry, instead of immunofluorescence.https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  5 Feb 2020Dear Dr. Fukai,Thank you for forwarding reviewer comments on our article “Assessment of ICAM-1 N-glycoforms in Mouse and Human Models of Endothelial Dysfunction”. We appreciate the reviewer’s constructive comments. In the revised manuscript, we have addressed the comments raised. Specific responses are provided below with edits in the text indicated by red font color.Thank youSincerelyRakesh Patel, PhDReviewer #1:The study by Regal-McDonald et al used PLA to investigate co-localization of specific N-glycan stuctures and ICAM-1 in mouse and human atherosclerotic arteries and human AVF of chronic kidney disease patients . The results demonstrated that hypoglycosylated and α-2,6-sialylated ICAM-1 N-glycoforms are present in atherosclerotic murine and human arteries and AVF patients. The results also indicated that HM-ICAM-1 levels positively correlated with macrophage burden in both murine and human atherosclerotic tissue. The study is timely but lacks functional information on the role of ICAM-1 N-glycoforms in atherosclerosis or AVF.1. The figures of the manuscript are very low resolution.  Author response: Image resolution has been increased. Al images are at 600dpi resolution per journal guidelines.2. It would strengthen the findings of the manuscript if the authors confirm the results obtained using the innominate arteries by looking at total, HM / hybrid, a-2,6-sialylated, and a-2,3-sialylated ICAM-1 in the atherosclerotic mouse aorta. Author response: We did not have access to mouse aortic tissue, but have now included new data from the left carotid arteries from ApoE-/- mouse fed high fa diet. These vessels are commonly used to interrogate atherosclerosis disease mechanisms; see revised Fig 2. 3. How were the lesion and non-lesion segments identified?Author response: Human lesions were characterized by a cardiovascular pathologist using the defining characteristics of fibrosis, calcification, and fatty deposits. Mouse lesion segments were identified by the presence of increased neointimal hyperplasia compared to non-lesion segments. 4. Fig. 2 and 3 legends state that n=5 experiments were performed for CD68 immunostaining. The corresponding bar graphs show only 3 experiments.Author response: We apologize for this oversight. The manuscript has been updated with additional experiments and n numbers corrected accordingly.5. What is the correlation coefficient for Fig. 4 studies? It seems there would be no correlation if only the closed symbols are taken into consideration. It is recommended to perform more experiments for the correlation studies.Author response: Additional replicates have been added and correlation analyses separated out into HFD-fed experiments and partial carotid ligation to promote clarity. 6. In the high fat diet model , there was atherosclerosis in the brachiocephalic artery and HM/hybrid, HM, a-2,6-sialylated and a-2,3-sialylated ICAM-1 were increased in lesion vs. non-lesion segments. If there is atherosclerosis in these mice but no correlation between CD68 and ConA or HHL puncta, the mechanisms regulating ICAM N-glycosylation may not be important for monocyte adhesion and macrophage accumulation.Author response: As explained above, additional experiments were performed. With these additional replicates and the separation of models in the correlation analysis, ConA and HHL had a significant positive correlation with CD68 staining in HFD-mice and partial carotid ligation.7. Type 2 human atherosclerotic lesions show no ICAM-1 expression. The type 2 lesions are characterized by the presence of lipid-laden macrophages according to Stary et al . These findings suggest that ICAM-1 (and its N-glycoforms) does not play a role at the early phase of monocyte transmigration in human atherosclerotic vessels.Author Response: We do not make the claim that ICAM-1 N-glycoforms play a role in early monocyte adhesion. However, the lack of ICAM-1 staining may also be due in part to detection limitations in our assay. We have included discussion of this point in the revised manuscript. 8. Fig. 6B legends n=10 is misleading. It seems only n=5 samples per group was involved in the analysis. The authors need to carefully check the legends for accuracyAuthor response: Additional data was added to manuscript and legends adjusted accordingly.9. This is an observational study and no functional experiments have been included to investigate the role of different ICAM-1 N-Glycoforms in the development of atherosclerosis or pathogenesis of AVF maturation failure. The manuscript would be strengthened by the addition of mechanistic data.Author response: Recently published work out of our lab demonstrates that different ICAM-1 N-glycoforms selectively recruit monocyte subsets under flow. Please see citation # 18 in the manuscript.10. The rational for including the AVF data in the manuscript is not entirely clear. The mechanisms, leading to endothelial dysfunction in AVF failure vs atherosclerosis is different.Author response: Mechanisms leading to AVF do indeed differ from atherosclerosis, but endothelial dysfunction as a result of turbulent blood flow is an underlying component of both diseases states. We wanted to test distinct ICAM-1 N-glycoforms were also present in other disease settings where vascular inflammation and turbulent blood flow are underlying features. This is also further discussed in the manuscript. Reviewer #2: This is a novel approach to assessment of ICAM-1 N-Glycoforms in mouse and human models of endothelial dysfunction. In this paper using the proximity ligation assay (PLA), authors assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis.1. To establish the distinct ICAM1-N-glycoforms in inflammatory disease in ivvivo authors solely used PLA assay. Indeed this assay has some limitation, however the PLA positive red puncta was not clear in all images throughout the manuscript. As this data represents the first study to demonstrating ICAM1-N-glycoforms invivo authors should provide convincing images with good quality pictures for PLA assay. Author response: Image resolution has been increased.2. There was no description for Figure 2F and 2G in result section. Author should mention about these two figures.Author response: We apologize for this oversight. The manuscript has been edited to include these figures in the results section.3. In result section, Figure 3A author described that total ICAM-1, HM / hybrid N-glycoforms were increased in the ligated left carotid artery (LC) compared to the control right carotid artery (RC). But this statement was not matches with the Figure 3A. It looks like total ICAM1 and α-2,6-sialylated ICAM1 expressed more than HM/hybride. Fig 3C quantification was not reflects the Fig 3A images. Author should be careful about this discrepancy.Author response: Analysis of PLA puncta is between paired LC and RC sections, not across lectins . Direct comparison between lectins is not possible as each have different binding affinities to their target epitopes- similar to how two different antibodies may bind with distinct affinities. The presented data is to show an increase in the ICAM-1 N-glycoforms in the ligated vessel compared to the non-ligated vessel (LC vs RC). We have clarified this point in the text. 4. In Fig 4 and Fig 6 show correlations between CD68 staining and HM / hybrid, HM and α-2,6- sialylated, ICAM-1 respectively. It was not clear how author made this correlation graph. Author should provide some direct evidence to prove this statement that CD68 expression higher in HM/hybrid not other ICAM1 N-glycoformes.Author response: Correlation studies were performed by measuring CD68 staining in the same  sections on which PLA staining was performed. In the case of HM and hybrid ICAM-1, CD68 staining increased as puncta increased, indicating a positive association between the two. Statistical analyses were performed using Pearson correlation.Reviewer #3: The current study showed correlation of high mannose (HM)-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms with macrophage burden in the lesions in human and mouse atherosclerosis, and in AVF maturation failure in hemodialysis patients. HM-ICAM1 N glycofoms and sialylated ICAM1 N-glycoforms were assessed by proximity ligation assay, while macrophage burden were assessed by CD68 staining. This is an extension of author’s previous studies. Findings are novel and important. There are several issues that should be addressed to make manuscript stronger.1) Overall, image quality except Figure 7A is poor. High resolution image will be needed, since this is a crux of current study. Author response: Image resolution has been increased.2) To support HM-ICAM1 N glycoforms and a-2,6 sialylated ICAM1 N-glycoforms in lesions, they need to be shown by western analysis as well. Author Response: By western blot analyses, we have shown previously that ICAM-1 immunoprecipitated from atherosclerotic lesions has a MWt close to 75KDa suggesting it is a HM-glycoform. Our published studies show that ICMA-1 with complex N-linked sugars has a MWt closer to 100KDa. This information is included in the revised discussion.3) In panel 2F, 3D, 6A, please show macrophage staining by immunohistochemistry, instead of immunofluorescence. Author Response: Immunofluorescence has been used extensively to assess CD68 staining in lesions, and per our experience there are no pros/cons in IF vs IHC for this marker. We have added an IgG staining control to Figure 6 to further verify specificity of the antibodies used for IF-based detection of CD68.AttachmentPLoS response final.docxSubmitted filename: Click here for additional data file. 28 Feb 2020Assessment of ICAM-1 N-Glycoforms in Mouse and Human Models of Endothelial DysfunctionPONE-D-19-20764R1Dear Dr. Rakesh Patel,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Introduction: Chronic exposure to the weightlessness-induced cephalad fluid shift is hypothesized to be a primary contributor to the development of spaceflight-associated neuro-ocular syndrome (SANS) and may be associated with an increased risk of venous thrombosis in the jugular vein. This study characterized the relationship between gravitational level (Gz-level) and acute vascular changes.Methods: Internal jugular vein (IJV) cross-sectional area, inferior vena cava (IVC) diameter, and common carotid artery (CCA) flow were measured using ultrasound in nine subjects  while seated when exposed to 1.00-Gz, 0.75-Gz, 0.50-Gz, and 0.25-Gz during parabolic flight and while supine before flight . Additionally, IJV flow patterns were characterized.Results: IJV cross-sectional area progressively increased from 12 (95% CI: 9–16) mm2 during 1.00-Gz seated to 24 (13–35), 34 (21–46), 68 (40–97), and 103 (75–131) mm2 during 0.75-Gz, 0.50-Gz, and 0.25-Gz seated and 1.00-Gz supine, respectively. Also, IJV flow pattern shifted from the continuous forward flow observed during 1.00-Gz and 0.75-Gz seated to pulsatile flow during 0.50-Gz seated, 0.25-Gz seated, and 1.00-Gz supine. In contrast, we were unable to detect differences in IVC diameter measured during 1.00-G seated and any level of partial gravity or during 1.00-Gz supine. CCA blood flow during 1.00-G seated was significantly less than 0.75-Gz and 1.00-Gz supine but differences were not detected at partial gravity levels 0.50-Gz and 0.25-Gz.Conclusions: Acute exposure to decreasing Gz-levels is associated with an expansion of the IJV and flow patterns that become similar to those observed in supine subjects and in astronauts during spaceflight. These data suggest that Gz-levels greater than 0.50-Gz may be required to reduce the weightlessness-induced headward fluid shift that may contribute to the risks of SANS and venous thrombosis during spaceflight. When a person stands upright on Earth, gravity pulls body fluids  toward their feet, resulting in approximately 70% of body fluids residing below heart level . During z). Further, we have recently documented IJV flow pattern changes that might contribute to an increased risk of venous thrombosis  and to describe how vascular parameters change at levels less than 1.00-Gz. Characterizing these physiological changes in response to varying Gz-levels is an important step in determining what Gz-level may be required to reverse weightlessness-induced fluid shifts to serve as a viable countermeasure during long-duration spaceflight. Further, this information might provide the basis for a prediction of whether Gz-levels experienced on the Moon and Mars will be sufficient to prevent SANS development during exploration missions.Reversing the headward fluid shift has been proposed as a method to relieve venous congestion associated with weightlessness to mitigate the risk of SANS and venous thrombosis. One approach for achieving this effect is to create artificial gravity by centrifugation . Howeverz-levels and hydrostatic gradients, including preflight supine (1.00-Gz supine), 0.25-, 0.50-, and 0.75-Gz while seated during parabolic flight, and 1.00-Gz seated during level flight between parabolas. Weightless parabolas (0-G) were not included in this parabolic flight campaign. Vascular measurements included internal jugular vein (IJV) cross-sectional area, flow characterization, and pressure; common carotid artery (CCA) cross-sectional area and flow; and inferior vena cava (IVC) diameter using ultrasound. Beat-to-beat finger blood pressure and heart rate from a three-lead ECG configuration  also were acquired. Study protocols were reviewed and approved by the French National Comité de Protection des Personnes and the NASA Johnson Space Center Institutional Review Board. This study was one of 11 experiments included in the parabolic flight campaign managed by Novespace, Inc.  as part of the first International Space Life Sciences Working Group Campaign in June 2018.This study characterized a set of vascular parameters at different Gz-levels  at 1.8-G, both lasting about 20 s. Accelerometer data measured in the cockpit of the plane by Novespace and collected by our group independently in the experiment area  were used to identify the respective partial gravity epochs within which data were analyzed. The air pressure in the cabin was maintained at approximately 600 mmHg (800 mbars) during the parabolas, which corresponded to an altitude of about 2,000 m. The temperature was controlled to be between 20 and 25°C.Each flight included 31 parabolas , with thPotential test subjects were identified by Novespace based upon selection criteria provided by the investigators. Subjects had to be between 25 and 55 years old to be similar in age to the astronaut corps , but theIn the week before the flights, subjects were screened by one of the investigators (DSM) to ensure adequate visualization of the relevant anatomy with ultrasound within the time constraints of each parabolic maneuver. Nine subjects  were identified, with three subjects participating in testing on each of 3 flight days. Subjects were 39 ± 6 years old , 171 ± 11 cm tall (157–187 cm), and weighed 65 ± 10 kg (50–85 kg). All subjects were received verbal instruction and written documentation regarding study protocols and encouraged to ask questions before providing written informed consent. Written informed consent also was obtained from all participants for the publication of any potentially identifiable images.All but one subject was administered an antiemetic  under the supervision of the flight medical doctor before boarding the airplane. Dosages administered ranged from 0.2 ml (0.05 mg) to 0.7 ml (0.175 mg), with the majority of the subjects receiving 0.5–0.7 ml (0.125–0.175 mg). Although oral scopolamine is associated with orthostatic hypotension in some individuals , no sympWithin 3 h before the flight, baseline measurements were acquired while the subjects were supine. A sonographer acquired ultrasound images  of the right IJV for off-line analysis of cross-sectional area, Doppler flow characterization , and estz-levels, and the sonographers were seated directly behind them, reaching around the subject to acquire IJV, IVC, and carotid images. Both the sonographer and the subject wore a lap belt to prevent excessive upward movements when in partial gravity. Subjects rested against a pad that extended above the level of their head, and the head was supported on the left side with an additional pad that allowed the subjects to relax their neck muscles. The same sequence of ultrasound images was acquired during each of the partial gravity conditions and during level flight, with the sonographer focusing on the acquisition of 1 parameter in each parabola. Sonographers acquired 1.00-G seated ultrasound images during level flight in the minute before the 1.8-Gz pull-up and acquired the partial gravity images from 5 s after the start of the partial gravity period until the end of the parabola. Heart rate and blood pressure were recorded continuously.During the flight, subjects were seated upright to experience the stressors associated with different Gz pull-up) and for periods of partial gravity from 5 s after achieving partial gravity until the pull-out. Mean arterial pressure was calculated as the average of the whole blood pressure waveform. Three separate ultrasound images were acquired for each target in each condition and stored for offline analysis. Two sonographers independently analyzed each image in a blinded fashion, and their results  were compared. When the inter-observer difference exceeded 10% for an IJV cross-sectional area measurement or a carotid measurement or if it exceeded 20% for an IVC diameter measurement, a third sonographer analyzed the image; the average of the two closest values were used for statistical analyses.Average values for heart rate and blood pressure were calculated for periods during level flight from 40 to 10 s prior to the hypergravity phase . The Cohen’s kappa associated these ratings was 0.95 (95% CI 0.84–1.00). Due to the near perfect agreement, the discrepant value from rater 1 (senior level sonographer), along with all other identical ratings, was used for this analysis.We characterized the venous blood flow within the IJV using a 1–4 grading system that incorporated direction and pattern of the Doppler signal . Continuz-levels and IJV pressure for all subjects while seated at 0.75‐ and 1.00-Gz were not of adequate quality for analysis. IJV pressure measurements are particularly difficult in the seated posture in 1.00-Gz, given that the IJV is largely collapsed in this condition. A subset of the IJV pressure data was determined to be acceptable by one of the investigators (DSM) for five subjects during 1.00-Gz supine and while seated at 0.25‐ and 0.50-Gz. No acceleration data were recorded by the experiment’s accelerometer on day 2, so accelerometer data from Novespace were analyzed for that day only.Data were not available for all conditions for some subjects due to technical or logistical difficulties. Beat-to-beat finger blood pressure data were not available for two subjects on day 1 and one subject on day 2. IJV cross-sectional area for one subject at all Gz-level. Separate linear regression models were constructed to quantify the association between each outcome  and G-level . Gz-level was modeled as a series of indicator variables, where 1.00-Gz (seated) was defined as the referent value. Model parameters were estimated using generalized estimating equations using an independence correlation structure (GEE-Ind) to account for repeated measurements within subject. Linear combinations of parameters were computed to estimate both main effects  and differences  along with 95% confidence intervals and p-values . A logistic regression model was constructed to quantify the relationship between IJV flow ratings  and G-level. Odds ratios (OR), 95% confidence intervals, and p-values were computed using GEE-Ind to summarize the comparison when comparing each partial gravity level to 1.00-Gz seated. IJV pressures were compared among subjects with both 0.50-Gz seated and 1.00-Gz supine conditions using a paired t-test. All analyses were performed using R 3.6.2 , and box/line plots] were computed to summarize parabolic flight by G R 3.6.2 .z, respectively. The mean Gz-levels measured across the 3 flight days were 0.25 ± 0.02, 0.50 ± 0.02, and 0.75 ± 0.02 Gz. The Gz-level during straight-and-level flight preceding each parabola was 1.00 ± 0.05 Gz  in all but one subject in each condition  became more apparent. Waveforms in four of nine, five of eight, and seven of eight subjects were scored as two at 0.50-Gz seated, 0.25-Gz seated, and 1.00-Gz supine, respectively. IJV flow scores were not different than 1.00-Gz seated at 0.75-Gz  and at 0.50-Gz , but IJV flow scores were significantly different than 1.00-Gz seated when compared to 0.25-Gz  and 1.00-Gz supine . Waveforms corresponding to grades 3 and 4 were not observed in our subjects during parabolic flight.During 1.00-Gondition . At lowez were not technically adequate, but data were available for six subjects during three conditions: preflight supine in 1.00-Gz and while seated at 0.25‐ and 0.50-Gz. Individual data represented in z supine to 0.25-Gz seated for two subjects and an increased IJV pressure in one subject. However, IJV pressure at 0.50-Gz seated (9.5 ± 3.4 mmHg) was lower than 1.00-Gz supine (19.1 ± 7.6 mmHg) for all five subjects .One of the investigators (DSM) visually inspected all IJV pressure measurements to ensure the technical quality of the imaging before analyses were performed. This approach determined that data collected while subjects were seated at 0.75‐ and 1.00-Gz, IJV cross-sectional area increases and IJV flow pattern becomes pulsatile as the G-level decreases during brief periods of partial gravity produced by parabolic flight. There are two important perspectives that can be derived from these results. First, our data suggest that the minimum Gz-level required to preserve hydrostatic conditions in the venous system of the upper body close to the upright 1.00-Gz posture, preventing IJV engorgement and changes in IJV flow patterns associated with the headward fluid shift in weightlessness, is greater than 0.50-Gz. Second, our data suggest that Gz-levels on the Moon (0.16-Gz) and Mars (0.38-Gz) would not be sufficient to prevent the headward distribution of venous blood that distends and changes the basic flow characteristics of the IJV. Future work will be required to verify that these observations during acute partial gravity are representative of the effects of chronic exposures  and while seated in 0-Gz , but decreased by ~50% at 0.38-Gz (Martian gravity).In the three gravity conditions in which technically sound measures of IJV pressure were acquired, it was apparent that IJV pressure decreases as Gengorged , as reflengorged . IJV presubjects ; IJV preImportantly, these data were collected during acute exposures, and thus, it is not clear what effects will be observed during chronic stays in weightlessness or partial gravity when IJV dimensions, pressures, and flow patterns would be chronically altered compared to the normal exposures in Earth gravity. For example, the IJV flow pattern was normal in 11 astronauts before spaceflight, transitioning from continuous forward flow when seated to pulsatile flow when supine, but the sustained headward fluid shift might promote progression to more abnormal flow patterns in some individuals. After ~50 and ~150 days of spaceflight, while most observations of IJV flow were pulsatile (11 of 21 observations), some were stagnant (7 of 21 observations) or reversed direction  or reverse flow (grade 4) at lower G-levels. Thus, from these data, it is not possible to ascertain whether partial gravity exposures would restore normal, forward, or pulsatile IJV flow once stagnant or reverse flow patterns have developed during prolonged weightlessness. In fact, in only three of the seven cases, in which stagnant or reverse flow was observed after ~50 and ~150 days of spaceflight was lower body negative pressure at 25 mmHg of decompression, a level of lower body negative pressure does not approximate 1.00-Gz levels of orthostatic stress (z) and Martian (0.38-Gz) may not be at a reduced risk of SANS and IJV thrombosis when in these partial gravity environments. Recently, This study provides evidence that countermeasures targeting the prevention of the weightlessness-induced headward fluid shift will need to produce hemodynamic effects in excess of that created by 0.50-Gc stress , effectic stress . Thus, az axis would be influenced by the different Gz-levels in the same way that they would be during an artificial gravity countermeasure in weightlessness and during habitation of low gravity environments. This posture, however, has the potential to exaggerate fluid shifts during parabolic flight, in comparison to the fluid shifts associated with steady-state condition during longer partial gravity exposures  during long-duration spaceflight and assessment of the cumulative response to prolonged habitation in a partial gravity environment are required to substantiate these assertions. Inflight monitoring of the caudal fluid shifts and the effectiveness of countermeasures will be helpful in optimizing the countermeasure prescription and efficacy.We report for the first time that IJV cross-sectional area increases and the IJV flow pattern becomes more pulsatile as the Ghttps://lsda.jsc.nasa.gov/).Requests to access the datasets should be directed to NASA’s Life Sciences Data Archive , editing of the manuscript, and approval of final draft. CM contributed to the study implementation (including engineering support and data collection), editing of the manuscript, and approval of final draft. JS contributed to the study design, editing of the manuscript, and approval of final draft. SSL contributed to the interpretation of results, editing of the manuscript, and approval of final draft. BM contributed to the interpretation of results, editing of the manuscript, approval of final draft, and secured funding. NM contributed to the statistical analyses, interpretation of the results, drafting and revision of the manuscript, and approval of final draft. LP-S and MS contributed to the study design, editing of the manuscript, approval of final draft, and secured funding. All authors contributed to the article and approved the submitted version.SMCL, DM, CM, SSL, BM, and NM were employed by KBR, under the Human Health and Performance Contract to NASA, during the performance of this study.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Increased violent events happen in the general hospitals in China and yet non-psychiatric departments do not have tools for violence-tendency screening.The current study developed and evaluated two Inpatient Psychological Experience Questionnaires (IPEQs) for the screening of violence-related six mental health disorders: : anxiety, depression and suicidality; Inpatient Psychological Experience Questionnaire-2 (IPEQ-2): paranoid personality disorder, emotionally unstable personality disorder and histrionic personality disorder). Two initial IPEQs (IPEQ-1: 37 items and IPEQ-2: 30 items) were developed and assessed by domain experts. Then 1210 inpatients were recruited and divided into three groups  for IPEQs item selection and evaluation. During the two-stage item selection, three statistical methods including Pearson’s correlation coefficient, exploratory factor analysis and item response theory were applied. For the item evaluation, Cronbach’s alpha coefficient, test-retest reliability, criterion-related validity and construct validity of the final questionnaires were measured.P < 0.01). Confirmatory factor analysis supported the validity of the final IPEQs (P < 0.05), and the model fit index met the criterion generally.Twelve items were selected for each IPEQs. Cronbach’s alpha coefficients were 0.91 and 0.78 for IPEQ-1 and IPEQ-2, respectively. Test-retest replication ratios were 0.95 and 0.87 for IPEQ-1 and IPEQ-2, respectively. Correlation coefficients between different disorders and their related-tools scores were  and  for IPEQ-1 and IPEQ-2, respectively and were significant (The IPEQs developed in this study could be effective and easy-to-use tools for screening inpatients with violence-intendancy in non-psychosomatic departments. In recent years, increased violent events were reported in the general hospitals of China , 2, whicHowever, the recognition rate of mental health disorders by non-psychiatric medical workers was very low in China . The curThere were two main factors making it difficult to employ mental health disorders screening: first, the lack of necessary knowledge or skills in non-psychiatric departments for mental-health and related personality disorder assessment ; second,To address these issues, in this study we made efforts to learn from the design of existing assessment tools, and also integrate the clinical experience used in China, and develop the two questionnaires according that showed effective and easy-to-use in Chinese patients. IPEQ-1 is for the screening of anxiety, depression and suicidality; and IPEQ-2 is for the screening of PPD, EUPD and HPD. This article reported on the development of the initial questionnaire items, selection of the final questionnaire items, and evaluation of two new IPEQs. The entire study flow diagram was shown in Fig. To develop and evaluate the proposed two IPEQs, we recruited 1210 participants, who were inpatients from different department of First Hospital of Shanxi Medical University . All inpatients met the following inclusion criteria: (1) age ≥ 18 years, (2) being ability to complete the questionnaires independently, and (3) willing to participate in this study. Inpatients were excluded if they were unable to complete the questionnaire. The 1210 participants were separated into three groups for item selection and evaluation, with the sample size of 160, 450 and 600, respectively. The sample size of first-stage was calculated according to the ratio of subjects to items , which was suggested as 5:1 in the pre-investigation stage ; and theThe first group was used for item pre-selection, which contains 160 inpatients recruited from July 2018 to August 2018. Specifically, 40 were from the psychiatric department, and 120 from non-psychiatric departments, including respiratory, neurology, traditional Chinese medicine, oncology and general surgery. The questionnaires with incomplete or biased answers were recognized as invalid and excluded. Finally, there were 149 inpatients completed IPEQ-1 and were included in the statistical analysis. For IPEQ-2, the number was 130.The second group was used for the item-revaluation based on the second-stage item selection, which contains 450 inpatients, recruited from non-psychiatric departments in August and September, 2018, from several departments respiratory, neurology, traditional Chinese medicine, oncology, general surgery, nephrology, hematology, geriatrics, cardiology, gastroenterology, endocrinology, rheumatology, plastic surgery, urology, orthopedics, otolaryngology, neurosurgery and gynecology. For IPEQ-1, there were 423 valid questionnaires collected, and the number for IPEQ-2 was 419.The third sample group was used for the evaluation of the proposed IPEQs. This group was composed of 600 non-psychiatric inpatients recruited from September 2018 to October 2018. There were 547 completed questionnaires collected for IPEQ-1and 523 for IPEQ-2.We present participants demographics of three sample groups in Table From a literature review, we designed two questionnaires for the screening of violence-related six mental health disorders: IPEQ-1: screening anxiety, depression and suicidality; IPEQ-2: screening PPD, EUPD and HPD.  , which iThe candidate item of IPEQs were reviewed by a panel of 16 senior experts that consisted of professors, clinicians and researchers in the field of psychology and psychiatry from 5 cities in China . These experts had more than 10-year experiences in the field. They rated each item in the pool as “strongly approved,” “revise slightly,” “revise moderately,” “revise entirely or delete,” or “delete”; and put forward specific suggestions for revision.Initially, 37 and 30 items were selected for IPEQ-1 and IPEQ-2, respectively  (including Pearson’s correlation coefficient (PCC) and exploratory factor analysis (EFA)) and item response theory (IRT).n = 160) were asked to answer all 67 questions of the two initial questionnaires , otherwise, it was excluded [In the first stage of item selection, the participants in the first sample group (excluded . Then EFexcluded .a) of each item [a < 0.40) [In addition, IRT was used to test the relationship between subjects’ disorders and their responses to questions by the discrimination  . Based on = 450) were required to answer the 18-item version of IPEQ-1 and 21-item version of IPEQ-2 from the first-stage item selection. The survey data were analyzed following the same statistical workflow (CTT and IRT) as used in the first stage item selection. IPEQs aimed to include the items that were not only efficient but also brief. The selection of an item was based on its consistency with primary symptoms of its corresponding disorder in ICD-10 and its statistical significance to the questionnaire. After the two-stage item selection, 12 questions were selected for both IPEQ-1 and IPEQ-2, which were used as the final version IPEQs  were used to evaluate the reliability and validity of both questionnaires. The internal disorder-specificity were measured by using Cronbach’s alpha coefficient (threshold ≥0.50) [P < 0.05 [The third group (d ≥0.50) . The staP < 0.05 .The efficacy of the propose questionnaires was evaluated by measuring the consistency between the results of using the proposed IPEQs and the three commonly used existing psychometric assessment-tools: 238 out of 600 were randomly picked to completed self-evaluation using GAD-7 , PHQ-9 , and PDQThe GAD-7 was a 7-item anxiety scale, that score ranged from 0 to 21. Cronbach’s alpha was 0.92 for the GAD-7. The PHQ-9 was a 9-item depression questionnaire, which the ninth item pertains to self-harm. The PHQ-9 score ranged from 0 to 27 and Cronbach’s alpha was reported by developers to be 0.89 and 0.86. The PDQ-4+ was a 107-item questionnaire, designed to assess the 12 personality disorders . The cutoff score of 4 or 5 was for screening of personality disorders. Cronbach’s alpha was 0.51 ~ 0.74 for the PDQ-4 + .P < 0.05; 2), root mean square error of approximation (RMSEA) < 0.08; 3), goodness-of-fit index (GFI) > 0.90; 4), adjusted goodness of fit index (AGFI) > 0.90; 5), normed fit index (NFI) > 0.90; 6), non-normed fit index (NNFI) > 0.90; and 7), comparative fit index (CFI) > 0.90 [The constructional validity was measured by evaluating the relation between each item and it corresponding disorder using confirmatory factor analysis (CFA), with criteria set as: 1), factor loading > 0.50 and ) > 0.90 .The feasibility of the IPEQs was evaluated by examining completion rate and completion time. When the questionnaire can be understood and completed by participants easily, it is considered acceptable feasibility.The statistical analysis was conducted by using SPSS software version 20.0, MULTILOG software version 7.03 and LISREL software version 8.70.The results of the statistical analysis for the two-stage item selection were provided in Table Some items were to be excluded based on statistical results. For example, item 29 in IPEQ-1 was excluded, because the correlation coefficient was < 0.40. Item 12 in IPEQ-1 was excluded, because it was showed weak correlation with its corresponding disorder in PCC. The correlation coefficient between item 12 and suicidality was 0.71, which was higher than correlation with anxiety (coefficient = 0.65). Item 18 in IPEQ-2 was excluded, because it was also demonstrated weak correlation with its corresponding disorder in PCC. The correlation coefficient between item 18 and HPD was 0.78, which was higher than correlation with EUPD (coefficient = 0.76). Item 23, item 30 and item 32 in IPEQ-1 and item 1, item 2, item 13 and item 16 in IPEQ-2 were excluded, because there was no correlation between these items and their corresponding disorder in EFA.Some items were to be excluded based on its consistency with primary symptoms of its corresponding disorder in ICD-10 in order to achieve two brief questionnaires. For example, item 5 was excluded because it was about physical symptoms. Item 15 was excluded because it had a lower factor loading compared to other items in depression in EFA, and its language expression was close to the item of suicidality.Eventually, for each of two IPEQs, 12 items passed the selection scrutiny and were used as final versions of the questionnaires. As seen in Supplementary Table P < 0.01). . Test-retest reliability analysis indicated that they showed strong stability (correlation coefficient > 0.70 and ). Table .Table 4P < 0.01), indicated acceptable criterion-related validity. . The values of GFI, NFI, NNFI and CFI were met the acceptance criteria.As seen in Table P < 0.05), except for items 1, 9 and 12. However, these three items were recommended for retention by the results of CTT and IRT analysis. The values of RMSEA, GFI, AGFI, NFI, NNFI and CFI were met the acceptance criteria.As seen in Table The completion rate for IPEQ-1 and IPEQ-2 were 91.17% (547/600) and 87.17% (523/600), respectively. The average completion time were 3.19 ± 3.09 min and 3.71 ± 3.39 min, respectively.China legislated against violence to medical workers . UnfortuP < 0.01). These three tools are rarely directly used by non-psychiatric departments for multiple reasons. First, these tools were specifically designed for the use by psychiatric department and non-psychiatric doctors and nurses usually lack professional training for the effective use of those tools. Second, these tools usually contain too many question items and inpatients usually are short of patience to answer [Compared with three commonly used tools for psychological-assessment tools, which are usually used by professionals in psychiatric departments, our IPEQs demonstrated strong consistence(o answer . For exaP < 0.05). These results showed, as simple as our IPEQs (12 questions only), it is effective in screening the proposed six mental health disorders, which were implicated to be associated with violence tendency [These two IPEQs were developed based on the ICD-10, experts’ opinions and relevant scale reviews. Three statistical methods  were considered in the two-stage item selection. Moreover, our statistical analysis showed that the items within each IPEQ showed high internal consistency  and strong stability . The final IPEQs also well-matched with the original IPEQs in terms of structure  for IPEQ-1 cut-off by the percentile of the questionnaire total score, according to literature reports , 49. ICDMedical workers have traditionally assessed violence risk on an individual basis when it has happened. It is important to screen for potentially violent inpatients according to the underlying psychiatric motive, such as impulsiveness, through relatively accurate predicable tool. Previous studies showed that there were positive association between six mental disorders  and violence –14. ManyThere are several limitations that we will addressed in future studies. First, all participants were from only one general hospital in Taiyuan, Shanxi Province. The proposed IPEQs should be validated in non-psychiatric departments and data should be collected for further evaluation of the tool. Second, we selected six mental disorders that were implicated to have strong association with violence. Adding other violence-related mental health disorders  for testing may improve the accuracy of the IPEQs. Third, we did not use golden standard in the cut-off study. A main future research would be to recruit participants with psychiatrists’ diagnoses, which will be suggested as golden standard to calculate sensitivity and specificity. In that way, we can improve the cut-off setting for IPEQs. Forth, our study only used three statistical methods to select items. Other statistical methods  Test combined with the parallel analysis in EFA ) may helWe developed and evaluated two IPEQs that demonstrated satisfactory reliability and validity. By using clinical symptoms or personality traits associated with violence-related mental health disorders, patients with violent tendencies would be screened. However, further studies are needed in different participants to revise and improve IPEQs. This initial study guaranteed further validation of the two proposed IPEQs in non-psychiatric departments of the general hospitals in China.Additional file 1: Supplementary Table 1. The final version of IPEQ-1. Supplementary Table 2. The final version of IPEQ-2. Supplementary Fig. 1. Test information and measurement error and matrix plot of item characteristic curves obtained from the 18 (second version) items of IPEQ-1 using IRT. Supplementary Fig. 2. Test information and measurement error and matrix plot of item characteristic curves obtained from the 21 (second version) items of IPEQ-2 using IRT


IL8 mRNA expression was determined by real-time quantitative PCR in the peripheral blood mononuclear cells from heparinized blood. The plasma IL-8 levels were assessed by enzyme-linked immunosorbent assay. The result shows that the IL8 mRNA expression in the H group tended to increase; however, in the CHD group, there was a significant decrease (p=0.0111) compared to the N group. The IL8 mRNA expression and the plasma levels in the CHD group were significantly lower than those in the H group (p < 0.05). A significant negative correlation between the IL8 mRNA (r = −0.499) or plasma IL-8 (r = −0.3875) expression and CHD progression was observed (p < 0.05). In conclusion, the transcriptomic and the phenotypic IL-8 expression decreased significantly in the Thai CHD patients who had continuously received statin-group medications compared to the H and N group participants. Therefore, IL-8 should serve as a feasible marker and could be used to evaluate the therapeutic effects of statins and illustrate the pathology of CHD treatment.The role of interleukin-8 (IL-8), a pivotal chemokine in atherogenesis and coronary heart disease (CHD) development, is diverse and remains unclear. This cross-sectional study investigates the association of the IL-8 expression in hyperlipidemia (H) and CHD patients who have been treated with statin cholesterol-lowering drugs while undergoing coronary artery bypass grafting treatment. Fifty-five Thai volunteers including 13 normal (N), 24 H, and 18 CHD patients were enrolled for the investigation. All the CHD patients had been treated continuously with statin cholesterol-lowering medications since the disease was diagnosed and were undergoing coronary bypass grafting approximately one month later. Therefore, the CHD group was representative of a pathogenesis improvement in CHD. The  Coronary heart disease (CHD) is one of the most significant vascular disorders in the world that is caused primarily by atherosclerosis complications. Major atherosclerosis disease mechanisms have been correlated to a high level of cholesterol, genetics, and the environment; however, the pathogenesis is still not understood entirely. Without treatment, atherosclerosis enhances occlusion of the arteries leading to ischemic heart disease and sudden myocardial infarction (MI) .γ can induce foam cell formation by increasing cholesterol retention through attenuating the expression of the ATP-binding cassette, subfamily A, as well as inducing the expression of acyl-CoA acyltransferase 1 that is involved in cholesterol esterification IL8 or CXor CXCL8 , and othor CXCL8 , emphyseor CXCL8 , periodoor CXCL8 , and sysor CXCL8 . In CVD,or CXCL8 –20.Although numerous studies have reported the atherogenic effect and the association of increased IL-8 expression with the development of cardiovascular disorders and CHD, the pivotal roles of IL-8 are still controversial. The anti-atherogenic effect of IL-8 on myocardial tissue repair was documented in MI –23, whilIL8 mRNA expression and the plasma IL-8 levels among healthy controls (N), hyperlipidemia patients (H), and CHD patients taking statin cholesterol-lowering medications while undergoing coronary bypass grafting in the CHD pathogenesis.In this cross-sectional study, we investigated the association of D-PBS , TRIzol® Reagent , IsoPrep , and the RNeasy total RNA kit  for RNA preparation were employed in this research. Real-time quantitative- (qRT-) PCR utilized primers that were designed according to GenBank sequences and based on previous studies , 26. TheThe design of this study is summarized in This study was approved by the RSU ethical review board (RSUERB2018-001), the Faculty of Tropical Medicine, Mahidol University (MUTM 2012-031-01), and Phramongkutklao Hospital (Q004q/55_Exp). Before enrollment, all the participants were informed of the aims and benefits of the study before completing an informed consent form.Five milliliter of heparinized blood was collected from each participant of the three groups. Two milliliter of plasma was stored at –70°C for the detection of IL-8 by ELISA. The packed blood cells were resuspended in D-PBS and used to isolate the peripheral mononuclear cells (PBMCs). These were separated by Isoprep gradient centrifugation (Robbins Scientific Corporation) as described previously . ApproxiLipid profiles including TC, triglyceride (TG), LDL cholesterol (LDL-c), and HDL cholesterol were determined by a biochemistry analyzer  at the clinical laboratory center of Phramongkutklao Hospital and/or by the Bangkok RIA Laboratory.IL8 mRNA was amplified by qPCR using the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase according to the manufacturer's instructions . Amplification was conducted in a Bio-Rad CFX Connect Real-Time System  using the following primers: cIL-8F (forward) 5′-GGCACAAACTTTCAGAGACAG-3′ and cIL-8R (reverse) 5′-ACACAGAGCTGCAGAAATCAGG-3′ [GADPH) was used as a housekeeping gene to normalize the relative expression of IL8 with the primers: G6F (forward) 5′-ACAGAGTGAGCCCTTCTTCAA-3′ and G6R (reverse) 5′-GGAGGCTGCATCATCGTACT-3′ [ΔΔCt method [The total RNA was isolated from the PBMCs using the RNeasy extraction kit (Qiagen) according to the manufacturer's instructions. The TCAGG-3′ ; glyceraGTACT-3′ . PCR cont method . The qRTμL) from the three groups and prediluted standards were added and incubated for 2 h at room temperature. Then, the wells were washed to remove any unbound material, and 100 μL of diluted detection antibody was added for 60 min at room temperature. After the unbound materials were removed, 100 μL of avidin-horseradish peroxidase conjugate was added for 30 min at room temperature. The bound enzyme was detected by adding 100 μL of substrate solution C to each well and the reactions were stopped by adding 100 μL of stop solution. The optical density was determined at 450 nm using the Tecan Sunrise OEM Remote Microplate Absorbance Reader . All the samples were assayed in triplicate. The IL-8 levels were calculated from standard curves.Levels of plasma IL-8 were determined by Human IL-8 ELISA MAX™ Deluxe  according to the manufacturer's recommendations. In brief, 96 wells were coated with a capture antibody and incubated overnight at 2–8°C. Plasma samples .Clinical data were presented as medians (upper and lower range limits). The p > 0.05). The CHD patients were significantly older than the participants in the N and H groups. The levels of TC (p < 0.0001) and LDL (p=0.0002) were significantly higher in the H group than in the N group, and the levels of “bad” lipids including TC (p=0.0028), TG (p=0.0183), and LDL (p=0.045) were significantly higher in the H group than in the CHD group. The CHD patients had a lipid baseline that was not significantly different when compared to N group participants, except for TG (p=0.0387).The patients' characteristics, clinical manifestations, and the healthy controls were compared between groups and summarized, as shown in IL8 mRNA expression levels are represented as relative expression (fold change) (IL8 mRNA expression in the CHD group was significantly lower than that in the N (p=0.0111) and H groups (p=0.0297). The IL8 mRNA expression tended to be higher in the H group than in the N group (p > 0.05).The  change) . The IL8IL8 pattern expression, the level of IL-8 was higher in the H group compared to the N (p > 0.05) and CHD (p=0.0210) groups. Levels did not differ significantly between the N and the CHD groups (p > 0.05).The levels of plasma IL-8 in all the groups are shown in IL8 mRNA  or the plasma IL-8 level  and CHD improvement. The IL-8 and other parameters did not show significant correlation.There was a significant negative correlation between This cross-sectional study used N, H, and CHD groups to illustrate atherogenesis development from healthy to atherosclerosis (moderate risk of CHD), consequent CHD, and the final improvement of plaques in coronary arteries. This is in accord with previous studies, which found that the reduction in the LDL-C level and the degree of anti-inflammation are inversely related to atherosclerotic progression , 30. We Although research into the functions of IL-8 has proved controversial, most previous studies reporting the contribution of the IL-8 expression to the progression of cardiovascular disorders involved both acute and chronic vascular inflammation –20. IL-8in vitro study proved that IL-8 was a powerful trigger for firm adhesion of monocytes to the vascular endothelium under flow conditions [In acute inflammation, IL-8 functions in chemotaxis and induces migration of neutrophils and macrophages to the tunica intima of the subendothelial space, sites of injury, or endothelial fatty streaks . Localiznditions . In addinditions .Statins are selective competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA reductase that interfere with mevalonate-derived compounds and lower cholesterol. Therefore, statins are used to treat hypercholesterolemia and are involved in the primary and secondary prevention of cardiovascular diseases. Rosuvastatin and atorvastatin are newly developed statins, and the former can lower LDL-C more effectively than the latter (reviewed in ). This sκB. Moreover, it has been found to reduce adhesion molecules and some cytokines including IL-8, IL-6, and MCP-1 [Salmonella infections [Pseudomonas pneumonia [Several previous studies found that statin drugs have played a role in the anti-inflammation of cardiovascular disorders , 43, 44.fections , Pseudomneumonia , and chrneumonia .The therapeutic effects of rosuvastatin and atorvastatin reduce bad lipids, decrease vascular inflammation, and result in the regression of coronary atherosclerotic plaques . In thisIL8 mRNA expression and the IL-8 plasma protein levels increased concordantly in the H group compared to healthy control. In contrast, we observed a significant decrease in the IL8 mRNA expression (p=0.0126) and the corresponding protein levels (p=0.0247) in CHD patients compared with the H group participants. We assume that the decrease in the IL-8 expression in the CHD group was caused by the action of cholesterol-lowering medications, as previously described by patients taking rosuvastatin and atorvastatin [In our cross-sectional study, we found that the vastatin . These rvastatin .Macaca mulatta) on a high-fat/high-cholesterol diet [A previous study has demonstrated that IL-18 was a potential predictive marker for atherosclerosis progression in SIV-infected rhesus monkeys (rol diet . Similarrol diet , 49.IL8 mRNA expression and IL-8 levels in cardiovascular diseases and coronary artery syndrome. IL-8 is mediated through various mechanisms to contribute to cardiovascular disorders [α, MCP-1, IL-1β, and IL-6, should be considered for future research [This study has several limitations. First, the study did not include CHD patients who had never been treated with statins or other cholesterol-lowering drugs. We lack some information from the patients' characteristics such as the detail of coronary graft bypass or clinical history and the progress of disease. However, reliable literature has mostly reported an increase in the isorders , 32–36. isorders . Fourth,This study found that both the transcriptomic and the phenotypic IL-8 expression increased in hyperlipidemia. In contrast, a significant decrease in the amount of IL-8 was observed in Thai CHD patients who took statin medication. Based on our findings and previous studies on the role of IL-8 in the pathogenesis of CVD, IL-8 may have the potential to be used as an alternative marker to evaluate the therapeutic effects of the statin drug group.


Work has shown that RTs for a SD saccade preceded by a MD saccade are longer than when a SD saccade is preceded by its same task-type, whereas the converse switch does not influence performance . Experiment 1 yielded a 28 ms and 8 ms unidirectional switch-cost pre- and post-exercise, respectively (ps < 0.001); however, the magnitude of the switch-cost was reduced post-exercise (p = 0.005). Experiment 2 involved a non-exercise control condition and yielded a reliable and equivalent magnitude unidirectional switch-cost at a pre- (28 ms) and post-break (26 ms) assessment (ps < 0.001). Accordingly, a single-bout of exercise improved task-switching efficiency and thereby provides convergent evidence that exercise provides a global benefit to the core components of executive function.Executive function includes the core components of working memory, inhibitory control, and cognitive flexibility. A wealth of studies demonstrate that working memory and inhibitory control improve following a single bout of exercise; however, a paucity – and equivocal – body of work has demonstrated a similar benefit for cognitive flexibility. Cognitive flexibility underlies switching between different attentional- and motor-related goals, and a potential limitation of previous work examining this component in an exercise context is that they included tasks involving non-executive processes . To address this issue, Experiment 1 employed a 20-min bout of aerobic exercise and examined pre- and immediate post-exercise cognitive flexibility via stimulus-driven (SD) and minimally delayed (MD) saccades ordered in an AABB task-switching paradigm. Stimulus-driven saccades are a standard task requiring a response at target onset, whereas MD saccades are a non-standard and top-down task requiring a response only  The n-back task entails a string of stimuli and requires that a participant indicate when a current stimulus matches a target from n-steps earlier in the sequence . The Stroop interference task is an exemplar measure of inhibitory control requiring that participants ignore the standard response of reporting a word name  and instead provide a non-standard response of reporting the (incongruent) ink color in which the word is written  and high-fit (VO2max > 49.2 ml/kg/min) complete a 30-min single bout of exercise (via treadmill at 60% of VO2max) with pre- and post-exercise task-switching efficiency assessed via alternating parity and size judgment tasks . In this task, an Arabic numeral was presented at the center of a monitor and was surrounded by a solid or dashed square. When a solid square surrounded the number, participants reported if it was above or below five, whereas for a dashed square an odd/even report was provided. High-fit – but not low-fit – individuals exhibited a 58 ms post-exercise improvement in task-switching efficiency. In turn, 2max) and used an odd/even and vowel/consonant judgment task (AABB paradigm) to examine cognitive flexibility. In particular, when a letter-digit pair was presented in the upper quadrant of a computer monitor participants performed an odd/even judgment task, whereas when a letter-digit pair was presented in the monitor’s lower quadrant they were asked to make a consonant/vowel letter judgment. The authors reported that their exercise manipulation did not influence task-switching efficiency and concluded that the paradigm may not provide a reliable framework for measuring exercise-related changes to cognitive flexibility.As mentioned previously, cognitive flexibility is a core component of executive function that reliably benefits working memory and inhibitory control – but not cognitive flexibility – may be explained in the context of transcranial magnetic stimulation, lesion and neuropsychiatric studies reporting that the components of executive function are interconnected but independent processes  and pre- and post-exercise cognitive flexibility was examined via stimulus-driven (SD) and minimally delayed (MD) saccades performed in an AABB paradigm . The oculomotor task used here is hands-free and does not entail the perceptual categorization of a stimulus. Moreover, the task has been shown to provide the resolution to detect subtle executive changes associated with prodromal cognitive decline . The standard nature of SD saccades is exemplified by the fact that such actions are performed upward of 150,000 times each day and are frequently referred to as reflexive in nature (after the target is extinguished (dz > 1.5) unidirectional switch-cost  may be immutable to a single bout of exercise. We also included a separate experiment wherein participants sat and rested on the cycle ergometer for a time equivalent to the exercise intervention. The second experiment was designed to determine whether a possible post-exercise change in task-switching efficiency was exercise-specific or related to a practice-related improvement.This research was approved by the Health Sciences Research Ethics Board, University of Western Ontario, and prior to data collection all participants read a letter of information, addressed any questions to the research team, and then signed a consent form. Further, this study was conducted in accordance with latest version of the Declaration of Helsinki with the exception that participants were not registered in a database.The recommended power for this type of research is 0.8 and based on our group’s previous work  it was eParticipants were instructed to refrain from rigorous exercise, alcohol, and caffeine 12 h prior to the study and were encouraged to get 7 or 8 h of sleep the night before their participation. All participants reported adherence to these requests. Data collection occurred between 9:30 and 10:30 am and participants were instructed to eat a normal breakfast and to hydrate prior to the study.max which was followed by a step-transition to a moderate intensity  for 20 min. A moderate intensity was used based on our group’s work showing that a continuum of moderate to very-heavy exercise intensities does not differentially influence a post-exercise benefit to inhibitory control  with a heart-rate monitor affixed to their chest . The heart rate monitor was used to ensure that participants exercised within their prescribed intensity. The exercise session included a 2.5 min warm-up at 50% of participant’s HR control . During Prior to and following the exercise session participants sat on a height adjustable chair placed in front of a tabletop (760 mm in height) with their head secured in a head-chin rest. A 30-inch LCD monitor  was used to present visual stimuli and was placed at participants’ midline and 550 mm from the front edge of the table. The gaze position of participants’ left eye was measured via a video-based eye-tracking system  sampling at 1000 Hz. Two additional monitors visible only to the experimenter provided real-time point of gaze information and trial-by-trial saccade kinematics . A nine-point calibration of the viewing space was performed prior to data collection and was confirmed via a follow-up validation . Computer events were controlled via MATLAB  and the Psychophysics Toolbox extensions (v 3.0)  includin2) and included a luminance matched central fixation cross  that appeared as green or red and open white target circles (163 cd/m2: 2.5° in diameter) 13.5°  and 17.5°  to the left and right of the fixation cross and in the same horizontal meridian. The color of the fixation cross indicated the nature of an upcoming trial. For one half of the participants, a green fixation cross indicated a response to target location at target onset , whereas the red fixation cross indicated a response to target location after it was extinguished . As a result, MD saccades required inhibition of a pre-potent response. For the other half of participants, the green and red fixation cross indicated a MD and SD saccade, respectively. Visual stimuli were presented on a black screen  and were performed at pre- and post-exercise assessments. Trials were equally divided into 80 task-repeat  and 80 task-switch  trials. The ordering of target location  was pseudo-randomized in one of four pre-determined trial sequences. The first trial was counterbalanced for task-type  and was excluded from data analyses because it was neither a task-switch nor a task-repeat saccade. In advance of a block of trials participants were provided an instruction screen indicating that a SD saccade required that they “Look to the target as accurately as you can as soon as it appears”, whereas the instruction for a MD saccade indicated “Look at the target Following the pre-exercise oculomotor assessment participants immediately started the exercise intervention. The post-exercise assessment was completed when participants’ heart rate was less than 100 beats per minute . Each oculomotor task required 15–17 min to complete . The timing and length of our post-exercise assessment was based on research  reportin2, respectively. Saccade offset was determined when velocity fell below 30°/s for 40 ms. Trials involving an eye blink (2%) were removed as were trials with a RT less than 50 ms , saccade duration (time from saccade onset to saccade offset), saccade gain amplitude  and associated within-participant standard deviations . Median RTs were computed given the positive skew of distributions see , whereasp = 0.461].Eighteen participants  from University of Western Ontario community volunteered for this study and were independent of the participants recruited in Experiment 1. Experiment 2 involved the same inclusion criterion and same instruction-set  as Experiment 1. The minimum and maximum GLETQ scores were 35 and 88, respectively  and did not reliably differ from Experiment 1 . In turn, MD saccade pre- and post-exercise difference scores did not reliably differ  and were within an equivalence boundary (TOST: t(17) = 2.72, p = 0.007).Our F = 100.67, p < 0.001, ηp2 = 0.86. F < 1.19, ps > 0.289, all ηp2 < 0.07. Saccade gain variability yielded a main effect of task, F = 17.47, p = 0.001, ηp2 = 0.51: values were less for SD  than MD saccades   than MD  saccades,  = 0.05) .F = 20.72, p < 0.001, ηp2 = 0.55, and a task by task-transition interaction, F = 19.58, p < 0.001. ηp2 = 0.53. t(17) = 5.81, p < 0.001], whereas MD task-switch and task-repeat trials did not reliably differ  and the TOST statistic indicated that MD task-switch and task-repeat trials approached a conventional equivalence boundary . t(17) = 0.94, p = 0.36], and the TOST statistic indicated that SD switch-costs were within an equivalence boundary .Results produced a main effect of task-transition, F = 77.69, ηp2 = 0.82: values were shorter for SD  than MD  saccades. Saccade gain did not reveal any reliable effects or interactions, all F < 2.06, ps > 0.16, all ηp2 < 0.11  = 17.45, p = 0.001, ηp2 = 0.50  increase in RTs for SD task-switch compared to task-repeat saccades and this contrast was associated with a large effect size (dz = 1.65). In turn, RTs for pre-exercise MD task-switch and task-repeat saccades did not reliably differ and a TOST statistic indicated that this comparison approached (but did not attain) a conventional level of statistical equivalence (p = 0.065). Accordingly, the pre-exercise RT findings indicate a reliable unidirectional switch-cost and is a result taken to evince that the top-down task-set necessary to produce a non-standard MD saccade engenders lingering neural activity that proactively delays the planning of a subsequent SD saccade  (othesis) .An important consideration of our proposal is that the RT difference between SD task-switch and task-repeat saccades reflects a switch-cost and is not related to a planning facilitation for task-repeat trials due to the consecutive presentation of a single trial-type . In a pu95% = 7) than their task-repeat counterparts, whereas MD task-switch and task-repeat saccades did not differ and were within an equivalence boundary. Thus, a unidirectional switch-cost was observed at the post-exercise assessment. Notably, however, magnitude of the switch-cost was reduced post-exercise and was independent of any pre- to post-exercise changes in saccade duration, gain or gain variability. Of course, on its own the pre- to post-exercise change in the magnitude of the switch-cost could be attributed to a practice-related improvement in our oculomotor task. For that reason, Experiment 2 involved a non-exercise control condition and produced a pre-  and post-break  unidirectional switch-cost that did not reliably differ and were within an equivalence boundary to Experiment 1’s pre-exercise switch-cost . Accordingly, the combined findings of Experiment 1 and 2 support the assertion that 20-min of aerobic activity improved task-switching efficiency and is a result we interpret to reflect an exercise-based improvement to cognitive flexibility.Post-exercise RTs for SD task-switch saccades were reliably longer  for the exercise-based improvement in task-switching efficiency. Some neuroimaging  studies have reported an improvement in task-dependent activity within parietal and frontal regions post-exercise e.g., , whereasircuitry , it is r2peak/max)  level of fitness may not demonstrate a comparable post-exercise benefit to cognitive flexibility. A second limitation is that the present work employed only a 20-min session at a moderate intensity. Although previous work by our group has shown a post-exercise benefit in oculomotor inhibitory control for 10- and 20-min sessions of aerobic exercise across a continuum of metabolically sustainable intensities ] to heavy intensity (50% of the difference between LT and VOeak/max)  it is poeak/max)  may showeak/max)  employedA single bout of moderate intensity exercise improved task-switching efficiency and was a result independent of a practice-related improvement in the oculomotor task used here. Accordingly, our results demonstrate that the SD and MD oculomotor task-switching paradigm provides the resolution necessary to detect post-exercise benefits to cognitive flexibility. Moreover, we believe that the present findings add importantly to the literature insomuch as they provide convergent evidence that a single bout of exercise benefits each core component of executive function.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Health Sciences Research Ethics Board, University of Western Ontario. The patients/participants provided their written informed consent to participate in this study.DS and MH conceptualized the study, contributed to the methodology, and investigated the study. MH wrote the original draft of the manuscript, acquised the funding, contributed to the resources, and supervised the study. DS wrote, reviewed, and edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.